Micropropagation of Pinus species
| dc.contributor.advisor | Van der Westhuizen, A. J. | |
| dc.contributor.advisor | Botha-Oberholster, A. M. | |
| dc.contributor.author | Jacoby, Angeline | |
| dc.date.accessioned | 2017-06-09T06:36:47Z | |
| dc.date.available | 2017-06-09T06:36:47Z | |
| dc.date.issued | 1999 | |
| dc.description.abstract | English: The aim of this study was to develop an effective protocol for the micropropagation of Pinus patuIa and P. radiata. Micropropagation procedures by means of somatic embryogenesis on solidified medium and in cell suspension cultures as well as organogenesis were investigated. In addition the possible relationship between phenolic, auxin and cytokinin content within cuttings and the tendency of these cuttings to root, were to be investigated. The cones of P. patuIa and P. radjata were collected for the somatic embryogenesis study, on a two weekly basis during the summer months of 1995 to 1997. Somatic embryonic cultures were initiated from the immature female gametophytes containing zygotic embryos. The embryonal suspensor mass (ESM) formed, was used as starting material for cell suspension cultures. Organogenesis included axillary and adventitious budding on hypocotyls and cotyledons respectively of young germlings deriving from seeds of open pollinated cones. Various techniques to sterilize the seeds were evaluated and it was found that 30% H202 (10 min.) proved most effective for P. patuIa, and 10% H202 (5 min.) was most effective for P. radiata. The initiation of somatic embryonic cultures was attempted on solidified modified Murashige and Skoog medium (MSG), Schenk and Hildebrandt (SH), Gresshof and Day (GO), Quoirin and Lepoivre (LP) and variations of the Douglas-fir Cotyledon Revised (OCR) media, each differing with regard to nitrogen sources and growth regulator composition. It was concluded that the most effective initiation media for P. patuIa were DCR1 and DCR5, and that DCR2 was most effective for P. radiata. Maintainance of the embryonic cultures was most successfully achieved for both species on ½ LP medium containing 3% maltose and no growth regulators. Maturation of P. radiata somatic embryos was achieved on solidified OCR2 medium supplemented with 1.3 rnql-¹ ABA, 30 gl-¹ glucose and 1% (mIv) activated charcoal. Attempts to mature P. patuIa embryos were unsuccessful. Embryonic cell suspension cultures were established in liquid GO, OCR, SH and ½ LP media. The best culture growth was achieved on ½ LP medium supplemented with 0,5 mql-¹ 2,4-0 and maltose as carbon source. Re-establishment of these cultures onto solidified ½ LP medium, supplemented with ABA, for further development and maturation was successful. Adventitious buds were induced on young (14 day old) cotyledons on nutrient (OCR) medium containing cytokinins (2 rnql-¹ BAP and 0.5 rnql-¹ Kin). In addition axillary buds were initiated on hypocotyls. A better success rate was obtained by axillary budding on hypocotyls than adventitious budding on cotyledons. Best elongation of the axillary buds was recorded on OCR medium containing no growth regulators. Rooting of these elongated shoots was subsequently successfully conducted on a GO medium supplemented with 0.5 rnql-¹ NMand 2 mgI-¹l IBA. An investigation on possible chemical markers of the rooting potential of cuttings was conducted on softwood cuttings of P. elliottii hybrids. The rooting percentage correlated inversely with the total phenolic content of the cuttings. According to TLC chromatograms for the separation of phenolic acids no special phenolic acid could be related to high or low rooting potential. Immunoassays were used to determine the endogenous auxin and cytokinin levels of cuttings. The rooting percentage correlated positively with the auxin concentration and negatively with the cytokinin concentration as expected. Results obtained in this study showed that the micropropagation of P. patuIa and P. radiata is feasible. These results contribute to a better understanding of micropropagation of Pinus species which has great potential for mass propagation demanded by forestry. | en_ZA |
| dc.description.abstract | Afrikaans: Die doel van hierdie studie was om 'n effektiewe protokol vir die mikropropagering van Pinus pafuIa en P. radiafa te ontwikkel. Mikropropagering by wyse van somatiese embriogenese op vaste medium en in selsuspensiekulture sowel as organogenese is ondersoek. Hierbenewens is die verband tussen fenol-, ouksienen sitokinieninhoud van steggies en die bewortelingstendens van steggies ondersoek. Die keëls van P. pafuIa en P. radiafa benodig vir die somatiese embriogenesestudie, is tweeweekliks gedurende die somermaande van 1995 tot 1997 versamel. Somatiese embriokulture is vanaf onvolwasse vroulike gametofiete wat sigotiese embrios bevat, geïnisieer. Die embrionale suspensie massa (ESM) wat gevorm het, is daarna in die selsuspensiekulture as moedermateriaal gebruik. Organogenese het oksel- en byknopontwikkeling op onderskeidelik hipokotiele en saadlobbe van jong kiemplantjies afkomstig van volwasse sade uit natuurlik bestuifde keëls behels. Verskeie tegnieke om saad te steriliseer, is beproef. Daar is bevind dat 30% H202 (10 min.) en 10% H202 (5 min.) die beste resultate vir P. pafuIa en P. radiafa onderskeidelik gelewer het. Daar is gepoog om somatiese-embriokulture op verskeie vaste media soos 'n veranderde Murashige en Skoog medium (MSG), Schenk en Hildebrandt (SH), Gresshof en Doy (GD), Quoirin en Lepoivre (LP) en variasies van die Douglas-fir Cotyledon Revised (DCR) media te inisieer. Die media verskil van mekaar t.o.v. stikstofbronne en die groeireguleerders. DCR1 en DCR5 was die effektiefste inisiëringsmedia vir P. pafuia en DCR2 was die effektiefste vir P. radiafa. Instandhouding van hierdie kulture is op ½ LP medium met 3% maltose en sonder enige groeireguleerders bereik. Veroudering van P. radiata somatiese embrio's tot volwassenheid was suksesvolop 'n vaste CR2 medium wat met 1.3 mgl-¹ ABA, 30 gr1 glukose en 1% (mIv) geaktiveerde koolstof verryk is. Pogings om P. patuIa embrios te verouder was onsuksesvol. Embriogenetiese selsuspensiekulture is in vloeibare GD, OCR, SH en ½ LP-media gevestig. Die beste kultuurgroei is op ½ LP medium verryk met 0.5 mgl-¹ 2,4-0 en maltose as koolstofbron behaal. Hervestiging van hierdie kulture is suksesvolop vaste ½ LP medium met ABA uitgevoer, vir verouderingsdoeleindes. Byknoppe is op jong (14 dae oue) saadlobbe op 'n sitokinienbevattende (2 mgl-¹ BAP and 0.5 mgl-¹ Kin) DCR-medium geïnduseer. Hierbenewens is okselknoppe op hipokotiele geïnisieer. 'n Hoër suksestempo is met die ontwikkeling van okselknoppe op hipokotiele as met die ontwikkeling van byknoppe op saadlobbe verkry. Die beste verlenging van die okselknoppe is op 'n DCR-medium met geen groeireguleerder waargeneem. Beworteling van die verlengde lote is suksesvolop 'n GD medium met O,5 mgl¹ NM en 2 mgl-¹ IBA uitgevoer. Ondersoeke om moontlike chemiese aanduidings van bewortelingspotensiaal van steggies te identifiseer is op sagtehoutsteggies van P. elliottii hibriede uitgevoer. Die totale fenoliese inhoud was omgekeerd eweredig aan die bewortelingspersentasie van die steggies. Volgens TLC-chromatogramme vir die skeiding van fenoliese sure is daar geen spesiale fenoliese sure wat met hoë of lae bewortelingspotensiaal geassosieer kan word nie. Die endogene ouksien- en sitokinienvlakke van die steggies is met behulp van immunoessaïeringsmetodes bepaal. Die bewortelingspersentasie het positief ooreengestem met die ouksienkonsentrasie en negatief met die sitokinienkonsentrasie gekorrelleer. Resultate van hierdie studie het bewys dat mikropropagering van P. pafuia en P.radiafa moontlik is. Hierdie resultate het tot 'n beter begrip van die mikropropagering van Pinus spesies bygedra. Mikropropagering het groot potensiaal vir massapropageering wat in aanvraag by die bosboubedryf is. | af |
| dc.description.sponsorship | FRD | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/6352 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Pinus radiata | en_ZA |
| dc.subject | Pinus patuIa | en_ZA |
| dc.subject | Micropropagation | en_ZA |
| dc.subject | Somatic embryogenesis | en_ZA |
| dc.subject | Embryonic cell suspension cultures | en_ZA |
| dc.subject | Tissue culture - Organogenesis | en_ZA |
| dc.subject | Axillary & adventitious budding | en_ZA |
| dc.subject | Phenolic | en_ZA |
| dc.subject | Immunoassays | en_ZA |
| dc.subject | Auxin & cytokinin | en_ZA |
| dc.subject | Pine -- Breeding | en_ZA |
| dc.subject | Pine -- Micropropagation | en_ZA |
| dc.subject | Dissertation (M.Sc. (Botany and Genetics))--University of the Free State, 1999 | en_ZA |
| dc.title | Micropropagation of Pinus species | en_ZA |
| dc.type | Dissertation | en_ZA |