Browsing by Author "Lennox, Sandra Janet"

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    The in vitro propagation of Veltheimia bracteata Harv. ex Bak
    (Stellenbosch : Stellenbosch University, 1990-12) Lennox, Sandra Janet; Watts, J. E.; De Lange, J. H.
    ENGLISH SUMMARY: The in vitro propagation of Veltheimia bracteata was studied to establish an efficient method of propagating unusual selections of V. bracteaca. particularly the yellow-flowered cultivar, "Lemon Flame", for their horticultural value. Shoots were induced from both bulb scale and leaf explants on growth media containing the inorganic salts of Murashige & Skoog ( 1962 ), as well as sucrose, kinetin and NAA. The recommended subculture period to promote shoot proliferation and avoid possible vitrification was 8 to 10 weeks. An advantage of leaf explants (against bulb scale explants ) is that the former can be easily decontaminated and the entire plant does not have to be destroyed. Initially, bulb segments were surface-sterilized by 70 % ethanol containing 0 ,1 % Tween 20, followed by immersion in 1 % NaOC1 containing 0 ,1 % Tween 20. The bulb scales were then separated, immersed in 1 % NaOC1 and 0,1 % Tween 20 and washed 3 times in sterile, distilled water. This procedure was later improved by a presterilization consisting of gentle scrubbing in distilled water containing Tween 20, after which the scales were immersed in 0, 3 % NaOC1 before being washed as described above . Leaves were washed under running water, scrubbed with 70 % ethanol containing 0.1 % Tween 20, sectioned, and then sterilized by being immersed in 0,5 % NaC1 containing 0. 1% Tween 20, followed by two rinses in sterile, distilled water. Two sizes of shoot tips, smaller than 1.0 mm in length, were excised from sterile cultures of V. bracteata and successfully cultured on BM supplemented with 10 mg. dm-³ kinetin and NAA. This was done as shoot-tip culture is one of the methods of producing "disease-free" plants, an important aspect of in vitro propagation where selected plants are rapidly multiplied. Shoots were more efficiently produced from the larger tips. The plants produced from the cultured shoot tips are presently under cultivation, to be compared with other micropropagated plants in order to identify differences which may be due to viruses or other diseases. The volume of culture containers did not have any effect on the foliar stomatal density of the plants grown in them. Neither did these densities differ significantly from those of plants grown under different conditions in the nursery. This indicated that the different containers would not have any significant effect on the acclimatization of in vitro cultured plantlets resulting from stomatal densities. Therefore, any of the three culture containers could be used for the micropropagation of V. bracteata. The propagation of V. bracteata by means of more conventional vegetative propagation methods, such as leaf cuttings, bulb scales or by scooping or scoring bulbs, was not nearly as efficient as in vitro propagation. However, the following methods, although slow, were identified as alternatives where tissue culture facilities are unavailable, or to be used in conjunction with tissue culture to propagate desirable nursery stock: scaling, with scales planted in sterilized , coarse sand, or scooping and treating the scooped bulbs with rooting hormone powder or kinetin. Shoot cultures of V. bracteata were rooted in vitro when cultured on BM supplemented with IBA (16 h light: 8 dark ) . Cultured shoots were also rooted extra vitrum in different soil mixtures, provided that these had been sterilized and treated with fungicide. Leaves torn from the base of micropropagated shoots could even be rooted extra vitrum in sand. Extra vitrum rooting is more economical than the labour intensive process of in vitro rooting. Cultured V. bracteata plants were successfully established in sand; soil and sand; bark and polystyrene; soil, bark and sand; as well as in sand and compost; but with less success in vermiculite. It is recommended that in vitro cultured shoots of V. bracteata be rooted extra vitrum in sterilized sand which had been treated with fungicide. Once rooted, plants should be transferred to a more fertile potting mixture to prevent roots from becomming brittle. Detailed procedures for the efficient isolation of protoplasts from bulb scale tissue of V. bracteata were developed. The optimal incubation period, concentrations of macerozyme and cellulase, medium molarity, suitability of osmotica and the influence of CaC1 2 on the isolation of protoplasts were investigated. The optimum medium contained ½ MS, 0,3 mol.dm-³ sorbitol, 0,1 mol.dm-³ CaC1 2 , 0,3 % cellulase and 0,05 % macerozyme. The inclusion of CaC1 2 in the isolation medium was essential for optimal protoplast yield as much lower protoplast densities were obtained in media without CaC1 2. Protoplasts isolated after an 8 h incubation period in the abovementioned isolation medium were purified by repeated centrifugation and resuspension in a washing medium containing 0,3 mol.dm-³ sorbitol and 0,1 mol.dm-³ CaC1 2. The protoplasts were collected after flotation on a denser sucrose pad and resuspended in culture medium to an optimal density of 2,0 x 10 5 protoplasts.g-¹. Optimal culture conditions are yet to be determined. The effect of different carbohydrates and the apparent toxicity induced by autoclaving carbohydrates in growth media should be taken into account in a Veltheimia bybridization programme. In the Veltheimia and Lachenalia plant selections studied, pollen germination and pollen tube growth were optimal on media containing sucrose and the poorest on galactose and fructose. For most of the plant selections studied, pollen germination and pollen tube length on media containing autoclaved carbohydrates were significantly lower than that on media containing unautoclaved carbohydrates. The addition of activated charcoal to media reduced the inhibitory effect of autoclaving.