Propagation of Romulea species.
Romulea is a genus with numerous attractive and endangered species with horticultural potential. This genus in the Iridaceae has its centre of diversity in the winter-rainfall zone of South Africa. This thesis uses ecophysiological and biotechnological techniques to investigate the physiology behind the propagation of some species in this genus. The ecophysiological techniques of soil sampling and analysis and germination physiology were used to determine the natural and ex vitro growth and development requirements of these plants, while biotechnological techniques are used to determine the in vitro growth and development requirements of these plants and to increase the rate of multiplication and development. Soil sampling and analysis revealed that R. monadelpha and R. sabulosa, two of the most attractive species in the genus, grow in nutrient poor 1:1 mixture of clay and sandy loam soil with an N:P:K ratio of 1.000:0.017:0.189 with abundant calcium. To investigate the physical properties of the seeds, imbibition rate, moisture content and viability of seeds were determined. The seed coat and micropylar regions were examined using scanning electron microscopy. To test for suitable stimuli for germination, the effect of temperature and light, cold and warm stratification, acid and sand paper scarification, plant growth promoting substances, deficiency of nitrogen, phosphorous and potassium, and different light spectra (phytochromes) on germination were examined. An initial germination experiment showed germination above 65% for R. diversiformis, R. leipoldtii, R. minutiflora and R. flava seeds placed at 15°C; while seeds of other species placed at 15°C all had germination percentages lower than 30%. More extensive germination experiments revealed that R. diversiformis and R. rosea seed germinate best at 10°C, R. flava seed germinates best when cold stratified (5°C) for 21 days and R. monadelpha germinates best at 15°C in the dark. Seeds of R. diversiformis, R. flava, R. leipoldtii, R. minutiflora, R. monadelpha and R. sabulosa seem to all exhibit non-deep endogenous morphophysiological dormancy while seeds of R. camerooniana and R. rosea appear to have deep endogenous morphophysiological dormancy. The suitability of various explant types and media supplementations for culture initiation was examined for various species of Romulea. Both embryos and seedling hypocotyls can be used for R. flava, R. leipoldtii and R. minutiflora in vitro shoot culture initiation. R. sabulosa shoot cultures can only be initiated by using embryos as explants, because of the lack of seed germination in this species. Shoot cultures of R. diversiformis, R. camerooniana and R. rosea could not be initiated due to the lack of an in vitro explant shooting response. Shoot cultures can be initiated on media supplemented with 2.3 to 23.2 M kinetin for all species that showed an in vitro response. The most suitable concentration depended on the species used. Some cultures appeared embryogenic, but this was shown not to be the case. A medium supplemented with 2.5 M mTR is most suitable for R. sabulosa shoot multiplication. BA caused vitrification of shoots in all the experiments in which it was included and is not a suitable cytokinin for the micropropagation of these species. The effect of various physical and chemical parameters on in vitro corm formation and ex vitro acclimatization and growth was examined. Low temperature significantly increased corm formation in R. minutiflora and R. sabulosa. A two step corm formation protocol involving placing corms at either 10 or 20°C for a few months and then transferring these cultures to 15°C should be used for R. sabulosa. When paclobutrazol and ABA were added to the medium on which R. minutiflora shoots were placed, the shoots developed corms at 25°C. This temperature totally inhibits corm formation when these growth retardants are not present. BA inhibited corm formation in R. leipoldtii. Corms can be commercialized as propagation units for winter-rainfall areas with minimum temperatures below 5°C during winter. Although an incident of in vitro flowering was observed during these experiments, these results could not be repeated. Although none of the corms or plantlets planted ex vitro in the greenhouse survived, a small viability and an ex vitro acclimatization experiment shows that the corms produced in vitro are viable. One embryo of the attractive R. sabulosa, produces 2.1 ± 0.7 SE shoots after 2 months; subsequently placing these shoots on a medium supplemented with 2.5 μM mTR for a further 2 months multiplies this value by 5.5 ± 1.3 SE. Each of these shoots can then be induced to produce a corm after 6 months. This means that 1 embryo can produce about 12 corms after 10 months or about 65 corms after 12 months (if shoots are subcultured to medium supplemented with 2.5 μM mTR for another 2 months). Embryo rescue can enable wider crosses within this genus. These results can be used for further horticultural development of species in this genus and their hybrids and variants.