Study On Somatic Embryogenesis And The Features Of Embryogenic Callus In Eucalyptus Urophylla×E.Grandis

Stem segments from sterile seedings of E. urophylla x E. grandis were cultivated on different media supplemented with different growth regulator combinations to induce embryogenic calli and somatic embryos. The Murashige and Skoog medium was more efficient for embryogenic calli formation than other media. Callus was formed at the surface of the explant in all MS medium with different growth regulator combinations. Approximately97%of explants cultivated on MS medium supplemented with0.5mg·L-1NAA and0.01mg-L"1TDZ formed embryogenic calli. Somatic embryogenesis was observed in callus in the presence of0.1mg-L"1NAA combined with0.01mg·L-1TDZ. The best somatic embryogenesis rate (16.7%) was obtained when explants were treated with5%sucrose for12h.Embryogenic calli and non-embryogenic calli were achieved from stem segment explants. The difference of embryogenic and non-embryogenic calli was studied through the observations of optical microscope of paraffin sections, the examinations of endogenous phytohormones, the evaluation of activities of antioxidant enzymes and the analyses of peroxidase isoenzyme by gradient polyacrylamide gel electrophoresis.Histological analysis of incubated explants revealed that embryogenic cells exhibited common features characteristic, i.e., they were small in size, had a large nucleus, and distributed in clusters. Non-embryogenic cells were differentiated,large and usually irregular, had a small nucleus and a large intercellular space.The levels of endogenous phytohormones were detected by HPLC. The result showed the contents of IAA, ABA and6-BA in embryogenic calli were higher than those in non-embryogenic calli while the content of GA3was higher in non-embryogenic calli. GA3may deteriorate the capacity of calli to differentiate. The addition of ABA was beneficial for embryogenic callus induction and somatic embryogenesis.Proteins and activities of antioxidant enzymes, including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), were evaluated in embryogenic and non-embryogenic calli in E. urophylla x E. grandis. In embryogenic calli, protein content and activities of CAT and SOD also showed significantly higher than non-embryogenic calli. According to the gradient polyacrylamide gel electrophoresis protein pattern, the strongest and weakest isoform bands of POD were detected in embryogenic and non-embryogenic calli, respectively. There were11POD isoforms detected in embryogenic calli while there are8in non-embryogenic. P3, P4and P10bands were present only in embryogenic calli. Thus, P3, P4and P10could be regarded as specific marker for embryogenic calli in E. urophylla×E. grandis. P3band were stronger than the others. It declared the metabolism associated with P3was active.