In vitro propagation of Pinus taeda via direct organogenesis from mature zygotic embryos

BARONE, J.; OBERSCHELP G., ; GONZALEZ A.M ; SANSBERRO P; LUNA C

La Plata

Congreso; Fourth International Conference of the IUFRO Working Party 2.09.02. Development and application of vegetative propagation technologies in plantation forestry to cope with a changing climate and environment. La Plata (Buenos Aires) Argentina. September 19-; 2016

IUFRO

Plant regeneration from in vitro culture of certain woody species requires look for complex techniques. Organogenesis and somatic embryogenesis have been considered the in vitro system of choice for the potential mass propagation of superior genotypes of forest species. Therefore, the aim of this work was to obtain an optimal combination of cytokinins on regeneration and production of buds from mature zygotic embryos of Pinus taeda. Mature zygotic seed embryos were cultured, after being scarified by agitation in H 2 O 2 30 vol. for 12 hours, disinfected by immersion in a 70° ethanol solution for 1 minute, then transferred to a 2.2% sodium hypochlorite aqueous solution added with 0.1% TRITON® and finally rinsed three times with sterile distilled water. The experiment used Murashige and Skoog basal medium (1962), with 30 g.L -1 sucrose and 6.5 g.L -1 Agar, in addition to 0.45, 5, 10, 13.6 µM of Thidiazuron (TDZ) and 0.45, 4.44, 9 y 13.31 µM of 6-benzylaminopurine (BA) alone or combined. The culture media were sterilized at 1.45 Kg.cm -2 for 20 minutes. The explants were cultured in laminar flow and incubated under light (116 µmol m -2 s -1 , 14 h photoperiod) and temperature (27±2 °C). Measurements were performed at 30, 45 and 60 days. A completely randomized experimental design was used, with five repetitions and an experimental unit of ten explants. The results were expressed as regeneration rate, oxidation rate of and number of buds per explant. The results were statistically analyzed by analysis of variance (ANOVA) and Tukey test (α=0.05). Even though bud regeneration was found associated with the presence of BA in the basal medium, there were no significant differences between concentrations of 4.44 µM and 13.31 µM with 56±8.94% and 44±15.2% respectively, although a concentration of 0.45 µM registered a lower regeneration of buds (10±6%). As to the number of buds per explants, it was higher in concentrations of 4.44 µM (9.39±2.25) and 13.33 µM BA (10.88±7.18). In the same way, the oxidation rate was not affected by the presence of this cytokinin. The highest concentrations of TDZ had a negative effect in regeneration and number of buds per explants; with 0.45 µM the regeneration rate was 40.67±11.63% and the number of buds per explants was 10.5±3, while with increased concentration both parameters decreased. The oxidation rate was affected too with the increase in the concentration of TDZ, from 34.6 ± 12.46% with 0.45 µM to 65 ± 10.49% 9 µM, and not being significant compared to 13.6 µM (63.33 ± 12.11%). As for the incubation time, the number of buds per explant was significantly superior at 45 days (10.11 ±2.8 buds/explant) compared to 30 days, while there was no significant difference at 60 days (9.1±3.5 buds/explant). Regarding the oxidation rate, it was significantly superior at 45 days (29.5±14.3 %), compared to 60 days of incubation (40.5±17.3%). In conclusion, 4.44 µM BA presented the highest regeneration rate, while the highest number of buds per explant was registered at 45 days of incubation, concurring with a lower oxidation rate.