The in vitro propagation of Veltheimia bracteata Harv. ex Bak

Lennox, Sandra Janet (1990-12)

Thesis (MSc)--Stellenbosch University, 1990.

Thesis

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.

AFRIKAANSE OPSOMMING: Die in vitro voortplanting van Veltheimia bracteata is bestudeer om 'n doeltreffende metode vas te stel waarmee besondere seleksies, veral die skaars "Lemon Flame" kultivar, vir hul tuinboukundige waarde voortgeplant kan word. Lote is geïnduseer vanaf bolskub- en blaareksplante op media bevattende die anorganiese saute van Murashige & Skoog ( 1962 ), asook sukrose, kinetien en NAA. 'n Subkultuur-periode van 8 to 10 weke word aanbeveel om lootver.neerdering te stimuleer en om moontlike lootvitrifisering te voorkom. Die voordeel van die gebruik van blaareksplante in plaas van bolskubbe is dat eersgenoemde makliker gedekontamineer kan word en dat die verkryging van eksplantmateriaal nie tot vernietiging van die bronplant lei nie. Bolskubbe is aanvanklik gesteriliseer deur dompeling van bolsegmente in 'n oplossing bevattende 70% etanol en 0,1% Tween 20, gevolg deur 'n oplossing bevattende 1 % NaOC1 en 0,1 % Tween 20 . Die bolskubbe is uitmekaar getrek, in 1% NaOC1 en 0,1 % Tween 20 gedompel en 3 keer in steriele gedistilleerde water gewas. Hierdie metode is later verbeter deur individuele bolskubbe in gedistilleerde water en Tween 20 te was vóór sterilisering in 0,3 % NaOCl soos hierbo aangedui. Blare is onder lopende wa ter afgespoel, in 70 % etanol en 0,1% Tween 20 afgevryf, opgesny, en daarna in 0,5% NaOC1 en 0,1 % Tween 20 gesteriliseer en twee keer in steriele gedistilleerde water gespoel. Lootpunte van twee groottes (kleiner as 1,0mm in lengte ) is uit steriele kulture van V. bracteata gedissekteer en suksesvol op BM bevattende kinetien en NAA, gekweek. Lootpunt-kulture is een van die metodes waardeur siektevrye plante verkry kan word en dit is 'n belangrike aspek van in vitro voortplanting waar geselekteerde plante vinnig vermeerder word. Lote is meer doeltreffend vanaf grater lootpunte as vanaf kleineres geproduseer. Die plante wat deur middel van lootpunt-kulture geproduseer is, word tans gekweek met die oog op die identifisering van groeiverskille tussen hierdie plante en plante wat in vitro gekweek is, as gevolg van die teenwoordigheid van virusse of ander patogene. Die volume van die houers waarin kulture van V. bracteata gekweek is, het nie verskillende huidmondjie-digthede tot gevolg gehad nie. Hierdie digthede het ook nie verskil van die van plante wat onder verskillende kwekerytoestande gekweek is nie. Dit het aangedui dat die verskillende kultuurhouers geen betekenisvolle invloed op die akklimatisering as gevolg van huidmondjie-digthede sou hê nie. Dus kan, binne perke, enige grootte kultuurhouer vir die mikropropagering van V. bracteata gebruik word. Alternatiewe metodes vir die vegetatiewe voortplanting van V. bracteata , byvoorbeeld deur middel van blaarsteggies, bolskubbe en uitgeholde of gesnyde bolle, is ook ondersoek. Hierdie metodes was nie so doeltreffend soos mikropropagering nie , dog die volgende metodes, alhoewel tydrowend, is geïdentifiseer as suksesvol: Die plant van "bolskubbe in gesteriliseerde growwe sand of vanuit uitgeholde bolle wat vooraf met Seradix 1 bewortelings-poeier of kinetien behandel is. Lootkulture van V. bracteata is op BM bevattende BA (16 h lig : 8 h danker ) in vitro bewortel. Lote is ook extra vitrum in verskillende grondmengsels wat vooraf gesteriliseer en met swamdoder behandel is, bewortel. Afgeskeurde blare vanuit kulture van V. bracteata kon selfs in sand extra vitrum bewortel word. Extra vitrum beworteling is meer ekonomies as die arbeid-intensiewe in vitro bewortelingsprosedure . In vitro-gekweekte plante is suksesvol in sand; grond en sand; bas en polistireen; grond, bas en sand asook in sand en kompos, maar minder suksesvol in vermikuliet, gevestig. Dit word aanbeveel dat in vitro-gekweekte lote extra vitrum in gesteriliseede sand wat met swamdoder behandel is, bewortel word. Bewortelde lote moet dan so gou as moontlik na 'n ryker grondmengsel oorgeplant word om te verhoed dat die wortels bros word. Prosedures vir die isolering van protoplaste vanaf bolskubbe van V. bracteata is ontwikkel . Die optimale inkubasie-periode, konsentrasies van maserosiem en sellulase, medium-molariteit, geskiktheid van osmotika en die invloed van CaC1 2 is bestudeer . Die mees doeltreffende medium is geïdentifiseer as een, bevattende ½ MS, 0,3 mol .dm-³ sorbitol, 0,1 mol .dm-³ CaC1 2, 0,3 % sellulase en 0,05 % maserosiem. Die insluiting van CaC1 2 in die medium was noodsaaklik vir 'n optimale protoplas -opbrengs omdat veel laer protoplast-digthede in media sander CaC1 2 verkry is. Protoplaste wat na 'n inkubasie periode van 8 h in bogenoemde medium geisoleer is, is herhaaldelik gesentrifugeer en geresuspendeer om die protoplaste te suiwer. Die wasmedium het 0,3 mol.dm-³ sorbitol en 0,1 mol.dm-³ CaC1 2 bevat. Protoplaste is op 'n digter sukrose laag gefloteer, versamel en daarna in kultuurmedium geresuspendeer tot 'n optimale digtheid van 2,0 x 10 5 protoplaste.g-¹. Optimale kultuurtoestande vir protoplaste moet nog vasgestel word. Die invloed van verskillende koolhidrate en die skynbare vergiftiging veroorsaak deur geoutoklaveerde koolhidrate in media moet in ag geneem word in 'n Veltheimia-kruisingsprogram . In die seleksies van Veltheimia en Lachenalia wat bestudeer is, was stuifmeel-ontkieming en die groei van stuifmeelbuise die beste op media met sukrose en swakste op media wat galaktose en fruktose bevat. Vir die meeste van die seleksies was stuifmeelontkieming en buisgroei op media met geoutoklaveerde koolhidrate betekenisvol laer as op media met ongeoutoklaveerde koolhidrate. Die byvoeging van geaktiveerde koolstof tot media het die inhiberende effek van outoklavering verminder.

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