Micropropagation of Pinus species

[摘要] English: The aim of this study was to develop an effective protocol for the micropropagationof Pinus patuIa and P. radiata. Micropropagation procedures by means of somaticembryogenesis on solidified medium and in cell suspension cultures as well asorganogenesis were investigated. In addition the possible relationship betweenphenolic, auxin and cytokinin content within cuttings and the tendency of thesecuttings to root, were to be investigated.The cones of P. patuIa and P. radjata were collected for the somatic embryogenesisstudy, on a two weekly basis during the summer months of 1995 to 1997. Somaticembryonic cultures were initiated from the immature female gametophytescontaining zygotic embryos. The embryonal suspensor mass (ESM) formed, wasused as starting material for cell suspension cultures.Organogenesis included axillary and adventitious budding on hypocotyls andcotyledons respectively of young germlings deriving from seeds of open pollinatedcones. Various techniques to sterilize the seeds were evaluated and it was foundthat 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 modifiedMurashige and Skoog medium (MSG), Schenk and Hildebrandt (SH), Gresshof andDay (GO), Quoirin and Lepoivre (LP) and variations of the Douglas-fir CotyledonRevised (OCR) media, each differing with regard to nitrogen sources and growthregulator 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 bothspecies on ½ LP medium containing 3% maltose and no growth regulators.Maturation of P. radiata somatic embryos was achieved on solidified OCR2 mediumsupplemented 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 ½LP media. The best culture growth was achieved on ½ LP medium supplementedwith 0,5 mql-¹ 2,4-0 and maltose as carbon source. Re-establishment of thesecultures onto solidified ½ LP medium, supplemented with ABA, for furtherdevelopment 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 additionaxillary buds were initiated on hypocotyls. A better success rate was obtained byaxillary budding on hypocotyls than adventitious budding on cotyledons. Bestelongation of the axillary buds was recorded on OCR medium containing no growthregulators. Rooting of these elongated shoots was subsequently successfullyconducted 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 cuttingswas conducted on softwood cuttings of P. elliottii hybrids. The rooting percentagecorrelated inversely with the total phenolic content of the cuttings. According toTLC chromatograms for the separation of phenolic acids no special phenolic acidcould be related to high or low rooting potential. Immunoassays were used todetermine the endogenous auxin and cytokinin levels of cuttings. The rootingpercentage correlated positively with the auxin concentration and negatively withthe 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 ofmicropropagation of Pinus species which has great potential for mass propagationdemanded by forestry.

[发布机构] University of the Free State