Micropropagation of Pinus species
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Date
1999
Authors
Jacoby, Angeline
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
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.
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.
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.
Description
Keywords
Pinus radiata, Pinus patuIa, Micropropagation, Somatic embryogenesis, Embryonic cell suspension cultures, Tissue culture - Organogenesis, Axillary & adventitious budding, Phenolic, Immunoassays, Auxin & cytokinin, Pine -- Breeding, Pine -- Micropropagation, Dissertation (M.Sc. (Botany and Genetics))--University of the Free State, 1999