The in vitro propagation of Veltheimia bracteata Harv. ex Bak

[摘要] 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 mediacontaining the inorganic salts of Murashige & Skoog ( 1962 ), as well assucrose, kinetin and NAA. The recommended subculture period to promoteshoot proliferation and avoid possible vitrification was 8 to 10 weeks. Anadvantage of leaf explants (against bulb scale explants ) is that the formercan be easily decontaminated and the entire plant does not have to bedestroyed.Initially, bulb segments were surface-sterilized by 70 % ethanol containing0 ,1 % Tween 20, followed by immersion in 1 % NaOC1 containing 0 ,1 % Tween20. 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 waslater improved by a presterilization consisting of gentle scrubbing indistilled water containing Tween 20, after which the scales were immersed in0, 3 % NaOC1 before being washed as described above . Leaves were washedunder running water, scrubbed with 70 % ethanol containing 0.1 % Tween 20,sectioned, and then sterilized by being immersed in 0,5 % NaC1 containing0. 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 fromsterile cultures of V. bracteata and successfully cultured on BMsupplemented with 10 mg. dm-³ kinetin and NAA. This was done as shoot-tipculture is one of the methods of producing disease-free plants, animportant aspect of in vitro propagation where selected plants are rapidlymultiplied. Shoots were more efficiently produced from the larger tips.The plants produced from the cultured shoot tips are presently undercultivation, to be compared with other micropropagated plants in order toidentify differences which may be due to viruses or other diseases.The volume of culture containers did not have any effect on the foliarstomatal density of the plants grown in them. Neither did these densitiesdiffer significantly from those of plants grown under different conditionsin the nursery. This indicated that the different containers would not haveany significant effect on the acclimatization of in vitro cultured plantletsresulting from stomatal densities. Therefore, any of the three culturecontainers could be used for the micropropagation of V. bracteata.The propagation of V. bracteata by means of more conventional vegetativepropagation methods, such as leaf cuttings, bulb scales or by scooping orscoring bulbs, was not nearly as efficient as in vitro propagation.However, the following methods, although slow, were identified asalternatives where tissue culture facilities are unavailable, or to be usedin conjunction with tissue culture to propagate desirable nursery stock:scaling, with scales planted in sterilized , coarse sand, or scooping andtreating the scooped bulbs with rooting hormone powder or kinetin.Shoot cultures of V. bracteata were rooted in vitro when cultured on BMsupplemented with IBA (16 h light: 8 dark ) . Cultured shoots were alsorooted extra vitrum in different soil mixtures, provided that these had beensterilized and treated with fungicide. Leaves torn from the base ofmicropropagated shoots could even be rooted extra vitrum in sand.Extra vitrum rooting is more economical than the labour intensive process ofin vitro rooting. Cultured V. bracteata plants were successfullyestablished in sand; soil and sand; bark and polystyrene; soil, bark andsand; 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 rootedextra vitrum in sterilized sand which had been treated with fungicide. Oncerooted, plants should be transferred to a more fertile potting mixture toprevent roots from becomming brittle.Detailed procedures for the efficient isolation of protoplasts from bulbscale tissue of V. bracteata were developed. The optimal incubation period,concentrations of macerozyme and cellulase, medium molarity, suitability ofosmotica and the influence of CaC1 2 on the isolation of protoplasts wereinvestigated. 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 inclusionof CaC1 2 in the isolation medium was essential for optimal protoplast yieldas much lower protoplast densities were obtained in media without CaC1 2.Protoplasts isolated after an 8 h incubation period in the abovementionedisolation medium were purified by repeated centrifugation and resuspensionin 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 andresuspended in culture medium to an optimal density of 2,0 x 10 5protoplasts.g-¹. Optimal culture conditions are yet to be determined.The effect of different carbohydrates and the apparent toxicity induced byautoclaving carbohydrates in growth media should be taken into account in a Veltheimia bybridization programme. In the Veltheimia and Lachenalia plantselections studied, pollen germination and pollen tube growth were optimalon media containing sucrose and the poorest on galactose and fructose. Formost of the plant selections studied, pollen germination and pollen tubelength on media containing autoclaved carbohydrates were significantly lowerthan that on media containing unautoclaved carbohydrates. The addition ofactivated charcoal to media reduced the inhibitory effect of autoclaving.

[发布机构] Stellenbosch University