Micropropagation of Pinus species

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.